Process for purification of a sialic acid from a fermentation broth
The present invention describes an efficient way to isolate a sialic acid from a fermentation broth. The sialic acid contained in the fermentation broth is produced by bacterial fermentation. The inventive process comprises a step of removing biomass from the fermentation broth, a step of subjecting the resulting solution to at least one of a cationic ion exchanger treatment and an anionic ion exchanger treatment and a step of removing salts after the ion exchanger treatment. The process can provide the sialic acid in spray-dried form as well as in form of sialic acid crystals.
1. A process for purification of a sialic acid from a fermentation broth, comprising:
removing biomass from a fermentation broth comprising a sialic acid, wherein a clarified solution is provided,
providing a purified solution by subjecting the clarified solution to
a cationic ion exchanger treatment with a cationic ion exchanger material, wherein the cationic ion exchanger treatment is performed under conditions in which the sialic acid passes the cationic ion exchanger material and is present in the flowthrough; and
an anionic ion exchanger treatment with an anionic ion exchange material, wherein the anionic ion exchanger treatment is performed under conditions in which the sialic acid passes the anionic ion exchanger material and is present in the flowthrough; and
removing salt from the purified solution by electrodialysis.
2. The process according to claim 1 , wherein the process does not comprise
i) a chromatographic separation; and/or
ii) a use of ethanol and/or ethyl acetate; and/or
iii) a step of eluting the sialic acid from a stationary phase with a solution comprising an organic solvent; and/or
iv) a use of a heavy metal; and/or
v) a step of heating the fermentation broth, the clarified solution and/or purified solution to a temperature of more than 45° C.
3. The process according to claim 1 , wherein the biomass is removed from the fermentation broth by centrifugation and/or filtration.
4. The process according to claim 1 , wherein in the cationic ion exchanger treatment, a strong cationic ion exchanger is used and/or in the anionic ion exchanger treatment, a strong anionic exchanger is used.
5. The process according to claim 1 , wherein the cationic ion exchanger treatment is performed to remove unspecific cations and replace them by specific cation H+ or Na+.
6. The process according to claim 1 , wherein the anionic exchanger step is performed to remove unspecific anions and replace them specific anion Cl− or OH−.
7. The process according to claim 1 , wherein conditions under which the sialic acid passes the anionic exchanger material and cationic exchanger material are established by adjusting the pH and/or salt concentration of the clarified solution.
8. The process according to claim 1 , wherein after the cationic ion exchanger treatment and the anionic ion exchanger treatment, the purified solution comprises the sialic acid, one or more colour-giving substances and salt.
9. The process according to claim 1 , wherein in the anionic ion exchanger treatment, an anionic exchanger material in the chloride form is used and/or in the cationic ion exchanger treatment, a cationic ion exchanger material in the hydrogen form is used.
10. The process according to claim 1 , wherein the clarified solution is subjected firstly to the cationic ion exchanger treatment and subsequently to the anionic ion exchanger treatment.
11. The process according to claim 1 , wherein the purified solution is concentrated.
12. The process according to claim 1 , wherein clarified solution and/or purified solution is concentrated
i) up to a concentration of ≥100 g/L of the sialic acid; and/or
ii) by nanofiltration at a temperature of <80° C., optionally <50° C., optionally 4° C. to 45° C., optionally 10° C. to 40° C., optionally 15 to 30° C., optionally 15 to 20° C.; and/or
iii) by reverse osmosis at a temperature of 20° C. to 50° C., optionally 30° C. to 45° C., optionally 35° C. to 45° C.; and/or
iv) at a pressure between >5 bar and <50 bar, optionally at a pressure between >10 bar and <40 bar, optionally at a pressure between >15 and <30 bar.
13. The process according to claim 1 , wherein the electrodialysis is an electrodialysis under neutral conditions or an electrodialysis under acidic conditions.
14. The process according to claim 1 , wherein after removing salt from the purified solution,
i) the amount of salt in the purified solution is <10% (w/w), optionally <5% (w/w), optionally <1% (w/w); and/or
ii) the conductivity is between 0.2 and 10.0 mS/cm 2 , optionally between 0.4 and 5.0 mS/cm 2 , optionally between 0.5 and 1.0 mS/cm 2 .
15. The process according to claim 1 , wherein the clarified solution and/or purified solution is subjected to discolouring by a treatment with activated charcoal.
16. The process according to claim 1 , wherein the sialic acid comprised in the clarified solution and/or purified solution is converted into sodium form.
17. The process according to claim 1 , wherein the purified solution is spray-dried.
18. The process according to claim 1 , wherein the purified solution is subjected to crystallisation.
19. The process according to claim 2 , wherein the process does not comprise a use of ethanol and/or ethyl acetate, and does not comprise the use of an organic solvent.
20. The process according to claim 5 , wherein if the unspecific cations are replaced by H+, the pH of the flowthrough is adjusted to a pH of 6 to 8 before performing a further treatment.
21. The process according to claim 20 , wherein the pH of the flowthrough is adjusted by addition of NaOH to the flowthrough.
22. The process according to claim 6 , wherein if the unspecific anions are replaced by Cl−, the pH of the flowthrough is adjusted to a pH of 6 to 8 before performing a further treatment.
23. The process according to claim 22 , wherein the pH of the flowthrough is adjusted by addition of NaOH to the flowthrough.
24. The process according to claim 7 , wherein the pH of the clarified solution is adjusted to a pH in the range of 6 to 8.
25. The process according to claim 8 , wherein the salt is NaCl.
26. The process according to claim 11 , wherein the purified solution is concentrated by nanofiltration and/or reverse osmosis.
27. The process according to claim 26 , wherein the purified solution is concentrated by nanofiltration, and wherein a nanofiltration membrane is used which has a molecular weight cut-off in a range of 100 to 200 kDa.
28. The process according to claim 12 , wherein clarified solution and/or purified solution is concentrated up to a concentration of ≥200 g/L of the sialic acid.
29. The process according to claim 28 , wherein clarified solution and/or purified solution is concentrated up to a concentration of ≥300 g/L of the sialic acid.
30. The process according to claim 15 , wherein the discolouring is performed
i) before or after a step of diafiltration and/or concentration of the clarified solution; and/or
ii) before or after a step of electrodialysis and/or diafiltration of the clarified solution.
31. The process according to claim 16 , wherein the sialic acid comprised in the clarified solution and/or purified solution is converted into sodium form by treating the purified solution with a strong cationic ion exchanger material in the sodium form.
32. The process according to claim 17 , wherein the purified solution comprises the sialic acid in the sodium form.
33. The process according to claim 18 , wherein the purified solution is subjected to crystallization by addition of acetic acid to the purified solution.
34. The process according to claim 33 , wherein sialic acid is provided in dihydrate crystal form.